The authors would like to acknowledge support from the Industry Alignment Fund Pre-Positioning (IAF-PP) funding (H18/01/a0/021 and H18/AH/a0/001) from A*STAR, Singapore.

<ol>
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P.J., A.B., R.L., and J.N. are employees of Thermo Fisher Scientific. A.L. and S.O. have no conflicts of interest.

In this work, we have shown the ability to close and semi-automate hMSC dissociation and wash and harvest on the bench using a counterflow centrifugation instrument. One of the critical steps in the entire workflow is making sure the tubings are connected according to the preset protocol defined in the counterflow centrifugation system protocol builder. The setup and operation are simple, and the time it took to process around 2 L of culture from a 10-layer flask from kit assembly to cell harvest was around 60 min. One of the limiting steps in this workflow is the fluid transfer from the multi-layered flask to the transfer bags connected to the counterflow centrifugation instrument. The high-flow single-use kit can only be run at a maximum flow rate of 165 mL/min, and this might be challenging for processing, for example, a 40-layer flask. To quicken the process of fluid transfer, external high-flow rate pumps can be used to transfer the trypsinized contents into a transfer bag first, followed by washing/concentrating and harvesting the cells from the transfer bag using the counterflow centrifugation system. Moreover, this protocol can also be applied for passaging cells from 4-layer to 10-layer multi-layered flasks. Further upstream, the counterflow centrifugation system could also be optimized for the washing of thawed hMSCs and direct harvest and medium formulation into multi-layered flasks to start the seed train. It should be noted that the minimum number of cells required to form the fluidized bed in the counterflow centrifugation chamber is approximately 30 million cells, and the maximum recommended volume to process per batch is 20 L.
Currently, the attachment of the custom tubing assembly to the multi-layered flask in the biosafety cabinet and the autoclaving of the parts of the components are not desirable in a cGMP setting. As an alternative, a custom gamma-sterilized tubing assembly could be outsourced to suppliers. Suppliers providing multi-layered flasks also offer the option of pre-fitting the flasks with desired tubing assemblies, including a 0.2 &#181;m filter, and gamma-sterilization of the entire outfit. This would ensure that the multi-layered flasks and attached tubing are truly closed, meaning the process could be completed on the bench in a Class C clean room setting.
This process utilizing the counterflow centrifugation system is not limited to adherent-based cultured cells in a multi-layered vessel and could be adapted to dynamic (stirred-tank or wave bioreactors) and static (gas-permeable) suspension-based cell expansion platforms. Specifically, for hMSCs expanded in 3D microcarrier cultures, protocols can be optimized on the counterflow centrifugation system to harvest, wash, and formulate the hMSCs dissociated from microcarriers. 
Overall, increasing interest in developing translational cellular therapies with improved process robustness and reliability has led to the development of closed, automated cell processing platforms. These systems are imperative, as they reduce the number of handling steps, prevent potential contamination by sterile connections, and reduce manufacturing costs by reducing labor and enhancing the effective use of clean room space21. In line with this, many of the cell therapy product developers who are seeking regulatory approval to translate their therapies are aware of the importance of closing the process and implementing full automation or semi-automation as early as the process development stage14,31,32. 
With the use of regulatory-friendly SFM XF medium, and along with ancillary reagents compliant with 21 CFR GMP Part 11 and international quality guidelines, this semi-automated process would be readily suitable for clinical manufacturing. We have shown the reproducibility of the closed process and maintenance of the quality of the WJ-MSCs. Improving the efficiency and safety of culturing adherent-based cells in multi-layered flasks would benefit not only the hMSC therapy field but also companies in cell line banking and adherent virus production.

Human mesenchymal stem cells (hMSCs) are a great candidate for clinical applications, both in tissue engineering and in cell therapies, given their therapeutic potential and high self-renewal potential to grow in vitro, which are critical for generating clinically-relevant dosages of cells1,2,3. According to ClinicalTrials.gov, there are over 1,000 clinical trials currently under investigation for various disease conditions4. Given the backdrop of increasing interest in using hMSCs, more clinical trials and market approvals are imminent in the near future5,6. However, the manufacturing of hMSCs has many inherent challenges in terms of batch-to-batch variability, the use of high-risk raw materials, concerns regarding contamination due to many open and manual processes, as the manufacturing involves multiple unit operations, higher labor costs, the cost of scaling out or scaling up, and regulatory hurdles6,7,8,9,10,11,12. These issues remain a significant barrier to current and future market access.
The development of closed, modular, automated manufacturing solutions and using low-risk ancillary reagents would address these challenges. This would also ensure consistent product quality, decrease the likelihood of batch failures due to human error, reduce labor costs, and improve process standardization and regulatory compliance, such as in terms of digital batch record-keeping8,12,13,14. To be able to obtain a clinically relevant dosage of cells, be it autologous or allogeneic, streamlined manufacturing that involves upstream cell expansion and downstream processing in a closed, automated manner is crucial.
For upstream hMSC expansion, the two most common manufacturing methods currently employed are scale-out (2D monolayer) and scale-up (3D microcarrier-based suspension system)15,16,17,18. The most traditional and widely adopted method for hMSC expansion is 2D monolayer-based culture due to the low production cost and ease of setup19.
Multi-layered flasks composed of flat surface trays stacked within a culture vessel are commonly utilized to scale out hMSC production. These systems typically come in 1-layer to 40-layer culture vessels20 and are handled manually inside biosafety cabinets. The processing steps during cell passaging and harvesting involve manually dispensing and decanting the expansion media, dissociation reagent, and wash buffer by pipetting or physically tilting the entire vessel. Besides, handling multiple units is challenging and time-consuming due to their sheer size and weight.
Subsequently, post-harvesting from multi-layered flasks, centrifugation for media exchange, cell wash, and volume reduction are essential steps across the entire cell manufacturing workflow21. Conventional benchtop centrifugation is a mostly open and manual process that involves a multitude of steps, such as transferring the cell suspension into capped tubes or bottles inside a biosafety cabinet, spinning down the cells, manually aspirating the supernatant, cell resuspension with the buffer, and repeated cell washes. This dramatically increases both the risk of contamination due to the opening and closing of the caps and the chances of losing the cell pellet during the manual aspiration/pipetting process22. In the context of handling multi-layered culture systems for adherent-based cells such as hMSCs, the operator would need to go through a laborious process of shuttling between the centrifuge and biosafety cabinet repeatedly and handling a heavy unit at the same time. These manual steps are laborious, pose risks in terms of human errors and contamination, and have to be conducted in a Class B clean room environment, which is costly23. In addition, the conventional manual centrifugation process is not scalable and could cause cellular shear and stress; thus, maximizing cell recovery, viability, and the wash-out efficiency of residual impurities are other major challenges22. Commercial cGMP scale manufacturing of cell therapies requires closed, modular automation solutions to reduce the risk of contamination, ensure consistent product quality, reduce labor and production costs, and increase process reliability24,25. Multi-layered flasks can be handled as a closed system by having a sterile 0.2 &#181;m filter in one of the ports to facilitate sterile gas exchange and a second port aseptically connected via connectors or tube-welded directly to an automated cell processing instrument for cell harvesting. We worked toward closing and automating most steps of WJ-hMSC passaging and harvesting by evaluating an innovative closed counterflow centrifuge intended for the manufacturing of cell, gene, or tissue-based products. This counterflow centrifuge also has the flexibility to perform a variety of cell processing applications, such as cell separation based on size, medium/buffer exchange, concentration, and harvesting for a variety of cell types8,26,27,28. The instrument uses a closed single-use kit that can be sterile-connected using tube welding or aseptic connectors to transfer bags or can be connected directly to any expansion platform of choice.
In this study, we designed a custom tubing assembly to allow closed sterile connections between the single-use counterflow centrifugation kit and the multi-layered flask. We optimized a protocol to enzymatically detach, wash, and harvest WJ-MSCs from the multi-layered flask in an entirely closed and semi-automated manner within a single run. The harvested WJ-hMSCs were characterized for purity (surface marker analysis) and potency (CFU-F, trilineage differentiation, and cytokine secretion profiles) to ensure that the final product met the critical quality attributes (CQAs) for lot release.

<table><tbody><tr><td>2L PVC transfer bag</td><td>TerumoBCT</td><td>BB*B200TM</td><td></td></tr><tr><td>Alcian blue solution, pH 2.5</td><td>Merck</td><td>101647</td><td></td></tr><tr><td>Alizarin-Red Staining Solution</td><td>Merck</td><td>TMS-008-C</td><td></td></tr><tr><td>APC anti-human CD73 Antibody</td><td>Biolegend</td><td>344015</td><td></td></tr><tr><td>APC Mouse IgG1, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend</td><td>400121</td><td></td></tr><tr><td>Bio-Plex MAGPIX Multiplex Reader</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counterflow Centrifugation System</td><td>Thermo Fisher Scientific</td><td>A47679</td><td>Gibco CTS Rotea Counterflow Centrifugation System</td></tr><tr><td>Crystal Violet</td><td>Sigma-aldrich</td><td>C0775</td><td></td></tr><tr><td>CTS (L-alanyl-L-glutamine) GlutaMAX supplement</td><td>Thermo Fisher Scientific</td><td>A1286001</td><td></td></tr><tr><td>CTS Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>A1285601</td><td>no calcium, no magnesium</td></tr><tr><td>CTS Recombinant Human Vitronectin (VTN-N)</td><td>Thermo Fisher Scientific</td><td>A27940</td><td></td></tr><tr><td>CTS TrypLE Select Enzyme</td><td>Thermo Fisher Scientific</td><td>A1285901</td><td></td></tr><tr><td>Custom tubing assembly</td><td>Saint-Gobain and Colder Product Company (CPC)</td><td>N/A</td><td>Gamma-sterilized 3/32&amp;rdquo; ID PVC line fitted with a sterile male MPC (1/8&amp;rdquo; barb) and sealed on the other end. Autoclave a short C-Flex line fitted with a sterile Cell Factory port connector on one end and a female MPC (3/8&amp;rdquo; barb) on the other. Connect the PVC and C-Flex lines in a biosafety cabinet</td></tr><tr><td>Emflon II capsule (0.2um filter)</td><td>Pall</td><td>KM5V002P2G100</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12662029</td><td>Mesenchymal stem cell-qualified, USDA-approved regions</td></tr><tr><td>FGF-basic</td><td>Thermo Fisher Scientific</td><td>PHG0024</td><td></td></tr><tr><td>FITC anti-human CD105 Antibody</td><td>Biolegend</td><td>323203</td><td></td></tr><tr><td>FITC anti-human CD45 Antibody</td><td>Biolegend</td><td>304005</td><td></td></tr><tr><td>FITC anti-human CD90 (Thy1) Antibody</td><td>Biolegend</td><td>328107</td><td></td></tr><tr><td>FITC Mouse IgG1, &amp;kappa; Isotype Ctrl (FC) Antibody</td><td>Biolegend</td><td>400109</td><td></td></tr><tr><td>Hi-Flow Single Use Kit</td><td>Thermo Fisher Scientific</td><td>A46575</td><td>Gibco CTS Rotea Hi-flow single-use kit, flow rate of 30 &amp;ndash; 165 mL/min</td></tr><tr><td>Multi-layered systems&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>140360 (4-layers); 140410 (10-layers)</td><td>Nunc Standard Cell Factory Systems</td></tr><tr><td>NucleoCounter NC-3000</td><td>Chemometec</td><td>NC-3000</td><td></td></tr><tr><td>Oil red O staining solution</td><td>Merck</td><td>102419</td><td></td></tr><tr><td>PDGF-BB</td><td>Thermo Fisher Scientific</td><td>PHG0045</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>PerCP anti-human CD34 Antibody</td><td>Biolegend</td><td>343519</td><td></td></tr><tr><td>PerCP Mouse IgG1, &amp;kappa; Isotype Ctrl Antibody</td><td>Biolegend</td><td>400147</td><td></td></tr><tr><td>ProcartaPlex Multiplex Immunoassays</td><td>Thermo Fisher Scientific</td><td></td><td>Custom 19-Plex panel: FGF-2, HGF, IDO, IL-10, IL-1RA, IL-6, IL-8, IP-10, MCP-1, MCP-2 , MIP-1&amp;alpha;, MIP-1&amp;beta;, MIP-3&amp;alpha;, PDGF-BB, RANTES, SDF-1&amp;alpha;, TGF&amp;alpha;, TNF-alpha, VEGF-A&amp;nbsp;</td></tr><tr><td>Sample port</td><td>Thermo Fisher Scientific</td><td>A50111</td><td>Gamma-sterilized leur sample port with 2 PVC lines attached</td></tr><tr><td>StemPro Adipogenesis Differentiation Kit</td><td>Thermo Fisher Scientific</td><td>A10070-01</td><td></td></tr><tr><td>StemPro Chondrocyte Differentiation</td><td>Thermo Fisher Scientific</td><td>A10071-01</td><td></td></tr><tr><td>StemPro Custom MSC SF XF Medium Kit (SFM XF medium)</td><td>Thermo Fisher Scientific</td><td>ME20236L1</td><td>Contains StemPro MSC SFM Basal Medium and Custom MSC SF XF Supplement (100x)</td></tr><tr><td>StemPro Osteogenesis Differentiation Kit</td><td>Thermo Fisher Scientific</td><td>A10072-01</td><td></td></tr><tr><td>T175 Nunc EasYFlask</td><td>Thermo Fisher Scientific</td><td>159910</td><td></td></tr><tr><td>T75 Nunc EasYFlask</td><td>Thermo Fisher Scientific</td><td>156472</td><td></td></tr><tr><td>TGF&amp;beta;1</td><td>Thermo Fisher Scientific</td><td>PHG9204</td><td></td></tr><tr><td>WJ MSCs&amp;nbsp;</td><td>PromoCell</td><td>(#C12971; Germany)</td><td>Human mesenchymal stem cells</td></tr><tr><td>&amp;alpha;MEM media</td><td>Thermo Fisher Scientific</td><td>12571063</td><td>With nucleosides</td></tr></tbody></table>

human mesenchymal stem cell processing for clinical applications using a closed semi-automated workflow

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

The WJ-hMSC master cell bank (MCB) post-thawing was maintained for three successive passages (p1-p4) in classic serum-containing medium to produce enough working cell banks (WCBs) for the experiments. The p4 WCBs were thawed and expanded in both serum-containing medium and SFM XF medium for three more passages (p4-p7) in T-175 flasks. The WJ-MSCs adapted well when expanded in SFM XF medium and were able to maintain stable proliferation similar to that in serum-containing medium (Figure 2A). However, the cells expanded in SFM XF medium exhibited slightly longer fibroblast-like spindle-shaped morphology, resulting in a slightly larger cell size (Figure 2B) of an average of ~17 &#181;m compared to ~15 &#181;m in serum-containing medium. In both medium conditions across the three passages, the WJ-hMSCs consistently reached their maximum cell density of ~2.3 x 104 cells/cm2 and a population doubling time of ~34 h (Figure 2C,D).
For large-scale WJ-hMSC expansion in a closed system, we carried out a seed train expansion of WJ-hMSCs first in a 4-layer flask and, subsequently, in a 10-layer multi-layered flask. At around 80%-90% confluency after 4 days of culture, we harvested 9.6 x 107 &#177; 0.9 x 107 and 2.3 x 108 &#177; 0.2 x 108 cells for the 4-layer and 10-layer stacks, respectively. A higher cell density of 3.6 x 104-3.8 x 104 cells/cm2 was reached compared to in the T-175 flasks, meaning the stacks allowed better cell expansion by up to seven-fold.
Further, the WJ-hMSCs expanded in 10-layer culture vessels were harvested directly using counterflow centrifugation. The sterile connection to the single-use kit was easy to establish for direct fluid transfer at a maximum flow rate of 165 mL/min using the instrument&#39;s peristaltic pump. The semi-automated cell harvesting process was achieved by first harvesting the cells using enzymatic dissociation, loading the cells into the counterflow chamber for volume reduction and concentrating, and then washing with wash buffer, which was around 3x the volume of the counterflow centrifugation chamber. Further, the washed cells were then concentrated and harvested to the desired harvest volume preset in the protocol. The processing steps used for the semi-automated cell processing were designed to emulate the manual harvesting workflow. We achieved a 10-fold volume reduction, resulting in the generation of cell concentrations as high as 5.3 million cells/mL. The protocol was able to achieve high cell recovery at ~98% and high cell viability at ~99% consistently for all three independent runs (Figure 3A-C).
We carried out extensive cell characterization assays to determine the critical quality attributes of the cell harvest using counterflow centrifugation compared to manual centrifugation. To test the identity of the WJ-hMSCs, cell surface markers were analyzed by flow cytometry. As shown in Figure 4A, the WJ-hMSCs harvested using both methods displayed characteristic surface marker profiles according to the ISCT regulations, positive expression of CD73, CD90, and CD105, as well as negative expression of CD34 and CD45. Next, to evaluate the clonogenic potential of the WJ-hMSCs, CFU-F assays were carried out. As shown in Figure 4B, cells harvested from counterflow centrifugation displayed a similar CFU-F potential compared with cells harvested by manual centrifugation (21% &#177; 1% vs. 20% &#177; 1%, respectively). Furthermore, as shown in Figure 4C, the post-counterflow centrifugation-harvested cells retained the ability to differentiate into adipocytes, osteoblasts, and chondrocytes similar to the cells in the manual centrifugation method. Lastly, we investigated 18 different cytokine secretion profiles of the cells using multiplex immunoassays. As shown in Figure 4D, the cells washed and concentrated using the post-counterflow centrifugation maintained cytokine secretion profiles, and the profiles were comparable to those of the sample taken before washing/concentrating the cells (pre-counterflow centrifugation).
Overall, we have demonstrated efficient hMSC expansion in an SFM XF culture system, and the cells washed and concentrated using the closed, automated counterflow centrifugation system yielded high cell recovery and viability post-wash and could maintain their phenotype and functionality. The closed semi-automated process developed in this study can deliver product quality consistency in terms of final WJ-MSC recovery, as evidenced from three independent runs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64707/64707fig01.jpg" /> 
Figure 1:&#160;High-flow single-use kit configuration and assembly for the harvest, washing, and concentration of hMSCs. (A) Kit diagram after the bags have been connected in line with the respective tubing. (B) The 10-layer multi-layered flask connected to the high-flow single-use kit with the custom tubing assembly. (C) Visualization of the stable fluidized cell bed formed in the counterflow chamber via the camera function enabled in the graphical user interface of the counterflow centrifugation software. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64707/64707fig02.jpg" /> 
Figure 2:&#160;Comparison of the cell morphology and expansion of hMSCs in serum-containing medium and SFM XF medium. (A) The representative cell morphology of hMSCs in classic serum medium and SFM XF medium. The SFM XF-expanded cells displayed a longer, spindle-shaped, characteristic fibroblast-like morphology, whereas the cells grown in serum-containing medium displayed a more flattened morphology. (B) The average MSC size between the serum-containing medium and SFM XF medium, measured by an automated cell counter (n = 3). It is clear to see that the SFM XF-expanded cells were generally larger than serum-expanded cells across different passages. The total cell yield in different passages (n= 3) (C) in terms of cells per culture surface area and (D) population doubling levels. Similar levels of cell yield were obtained between the SFM XF medium and serum-containing medium across different passages. Data are expressed as the mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64707/64707fig03.jpg" /> 
Figure 3: Characterization of cells processed using the counterflow centrifugation system. (A) Total viable cells before and after the washing and concentration. (B) A 10x volume reduction was achieved post-counterflow centrifugation processing. (C) Total recovery and viability of the cells. Data are averaged over three biological replicates (n = 3) of washing and concentration runs. Data are expressed as mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64707/64707fig04.jpg" /> 
Figure 4: Critical quality attributes analysis. (A) Representative data from flow cytometry. (B) Representative images showing the total CFU. (C) Representative microscopic images of the trilineage differentiation. (D) Cytokine expression analysis results before and after processing the cells on the counterflow centrifugation system (n = 3). Data are expressed as mean &#177; standard deviation. <a href="https://www.jove.com/files/ftp_upload/64707/64707fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequence of the hMSC harvest by trypsinization, washing, and concentration protocol on the counterflow centrifugation system, including the initial priming steps. <a href="https://www.jove.com/files/ftp_upload/64707/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow at JoVE.com

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market ...

human-mesenchymal-stem-cell-processing-for-clinical-applications

Research

JoVE Journal

Biochemistry

1.7K Views.  Agency for Science, Technology and Research (A STAR). Benchtop centrifugatie is een open en handmatig proces dat uitdagend is om op te schalen. Ons protocol laat zien hoe de dissociatie, het wassen en oogsten van mesenchymale stamcellen, of MSC's, in één keer kan worden gesloten en geautomatiseerd. De installatie is eenvoudig.Het hele proces kan buiten de bioveiligheidskast worden uitgevoerd in een klasse cGMP-faciliteit. Gesloten geautomatiseerde celverwerking vermindert handmatige verwerkingsstappen die risicogevoelig kunnen zijn en c...

Video: Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Developmental Biology

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Bioengineering

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Medicine

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

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